Methods for producing L-isoleucine

ABSTRACT

The present invention relates, in general, to the over-production of L-isoleucine by nonhuman organisms. More specifically, the present invention relates to methods for producing L-isoleucine comprising: (a) growing a transformed nonhuman organism under conditions that provide for synthesis of L-isoleucine, wherein the nonhuman organism comprises one or more copies of a transgene comprising at least one nucleotide sequence encoding catabolic threonine dehydratase; wherein the L-isoleucine is synthesized by the transformed nonhuman organism, the synthesis being greater than that of the corresponding non-transformed nonhuman organism; and (b) recovering the L-isoleucine from the culture medium in which the transformed nonhuman organism was cultured.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit to the filing dates of U.S. ProvisionalApplication No. 60/142,071, filed Jul. 2, 1999; and U.S. ProvisionalApplication No. 60/205,212, filed May 18, 2000; each of which is hereinincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates, in general, to the field of production ofamino acids. More specifically, the present invention relates to theover-production of isoleucine by nonhuman organisms.

2. Related Art

Corynebacteria have a long history of use in the industrial productionof amino acids, which are used as food additives (most notably lysineand other essential amino acids) and as flavor enhancers (monosodiumglutamate, or MSG). The overall global market for amino acids as animalfeed additives is estimated to be worth more than $2 billion and totalsabout 700,000 metric tons of material. Lysine and methionine account foran overwhelming majority of the market, which also includes lower-volumeproducts like threonine (Lessard, P. A., et al., “Corynebacteria,Brevibacteria,” in The Encyclopedia of Bioprocess Technology:Fermentation, Biocatalysis and Bioseparation, John Wiley & Sons, NewYork, N.Y. (April 1999), Volume 2, pp. 729-740). The current productionof isoleucine is less than 400 metric tons per year. This amino acid iscurrently used as a constituent of infusions and special dietaryproducts. As with other amino acids the demand for isoleucine isincreasing and its industrial production is expected to open upadditional markets as an animal feed additive. Due to the tight controlof isoleucine biosynthesis in bacteria, this amino acid is still in partproduced commercially by direct extraction from protein hydrolysates.

The Gram positive Corynebacterium glutamicum is currently used inindustry to produce over 100 grams of lysine per liter of culture. Theflux of carbon through metabolic pathways can be diverted from theproduction of lysine to make other related amino acids by the processesand methods of metabolic engineering. Traditional metabolic manipulationinvolved random mutagenesis and screening for the desired changes inphysiology, but transformation and genetic manipulation tools have beendeveloped in the last ten years to allow more direct engineering ofspecific pathway elements in Corynebacterium (Jetten, M. S. M., et al.,“Molecular organization and regulation of the biosynthetic pathway foraspartate-derived amino acids in Corynebacterium glutamicum,” inIndustrial microorganisms: basic and applied molecular genetics, Baltz,R. H., et al., eds., Am. Soc. Microbiol., Washington, D.C. (1993), pp.97-104; Lessard, P. A., et al., “Corynebacteria, Brevibacteria,” in TheEncyclopedia of Bioprocess Technology: Fermentation, Biocatalysis andBioseparation, John Wiley & Sons, New York, N.Y. (April 1999), Volume 2,pp. 729-740).

L-Isoleucine belongs to the aspartate-derived family of amino acids, asdo lysine, homoserine, threonine and methionine. The enzymes thatsynthesize this family of amino acids have been well characterized inCorynebacterium, as has their regulation (FIG. 1). The first importantregulatory point in the production of isoleucine by C. glutamicum is theend-product inhibition of the first committed enzyme, threoninedehydratase (E.C. 4.2.1.16), encoded by the gene ilvA. Overproduction ofisoleucine has been accomplished by introducing excess threoninedehydratase (encoded by ilvA) into threonine producer strains (Colón, G.E., et al., Appl. Microbiol. Biotechnol. 43:482-488 (1995)). Threoninedehydratase is normally feedback inhibited by isoleucine. Mutantderivatives of threonine dehydratase with reduced sensitivity toisoleucine provided an additional dividend in this isoleucine productionsystem (Hashiguchi, K., et al., Biosci. Biotechnol. Biochem. 61:105-108(1997); Morbach, S. et al., Appl. Env. Microbiol. 61:4315-4320 (1995)).Despite these gains, it appears that amino acid export has been aserious limitation to the effectiveness of amino acid production (Kelle,R., et al., J. Biotechnol. 50:123-136 (1996)).

Work on artificial enzyme evolution has shown that it is difficult tosubtly alter a task for which an enzyme was specifically evolved, whileit is easier to coopt an enzyme for a completely new task (Benner, S.A., Chem. Rev. 89:789-806 (1989)).

One such alternative enzyme might be the catabolic threoninedehydratase, also called biodegradative threonine dehydratase, (E.C.4.2.1.16), which is also known as threonine deaminase. This threoninedehydratase is produced in E. coli cells when the organism is grownanaerobically in a medium containing high concentrations of amino acidsand no glucose (Umbarger, H. E. & Brown, B., J. Bacteriol 73:105-112(1957)). In contrast to the threonine dehydratase encoded by ilvA, ananabolic threonine dehydratase, the enzyme encoded by tdcB in E. coli isinsensitive to inhibition by L-isoleucine and is activated by adenosine5′-monophosphate. The tdcB gene from E.coli has already been cloned andsequenced (Goss, T. J. & Datta, P., Mol. Gen. Genet. 201:308-314(1985)).

In the past, overproduction of isoleucine has been accomplished byintroduced excess threonine dehydratase encoded by ilvA, an anabolicthreonine dehydratase, into threonine producer strains (colón, G. E., etal., Appl. Microbiol. Biotechnol. 43:482-488 (1995)). Although theconventional methods have considerably enhanced the production ofisoleucine, the development of a more efficient, cost-effectivetechnique is required in order to meet increasing demand forL-isoleucine.

BRIEF SUMMARY OF THE INVENTION

It is a general object of the invention to provide a method forover-producing L-isoleucine.

It is a specific object of the invention to provide a method forproducing L-isoleucine comprising growing a transformed nonhumanorganism under conditions that provide for synthesis of L-isoleucine,wherein said nonhuman organism comprises one or more copies of atransgene comprising at least one nucleotide sequence encoding catabolicthreonine dehydratase, wherein said L-isoleucine is synthesized by saidtransformed nonhuman organism, said synthesis being greater than that ofthe corresponding non-transformed nonhuman organism, and recovering saidL-isoleucine from said culture medium in which said transformed nonhumanorganism was cultured.

It is a specific object of the invention to provide a method forproducing L-isoleucine comprising growing an L-isoleucine producingnonhuman organism comprising one or more copies of a transgenecomprising at least one nucleotide sequence encoding catabolic threoninedehydratase, wherein said nonhuman organism synthesizes L-isoleucine,said synthesis being greater than that of a corresponding nonhumanorganism which does not comprise one or more copies of a transgenecomprising at least one nucleotide sequence encoding catabolic threoninedehydratase, and recovering said L-isoleucine from said culture media inwhich said L-isoleucine producing nonhuman organism was cultured.

It is another specific object of the invention to provide a transformednon-human organism, wherein said transformed nonhuman organism is anL-isoleucine producing nonhuman organism, comprising one or more copiesof a transgene comprising at least one nucleotide sequence encodingcatabolic threonine dehydratase, wherein said transformed nonhumanorganism synthesizes L-isoleucine, said synthesis being greater thanthat of the corresponding non-transformed nonhuman organism.

It is another object of the invention to provide an L-isoleucineproducing nonhuman organism comprising one or more copies of a transgenecomprising at least one nucleotide sequence encoding catabolic threoninedehydratase, wherein said nonhuman organism synthesizes L-isoleucine,said synthesis being greater than that of a corresponding nonhumanorganism which does not comprise one or more copies of a transgenecomprising at least one nucleotide sequence encoding catabolic threoninedehydratase.

It is another specific object of the invention to provide a transformedorganism, Corynebacterium glutamicum ATCC21799 comprising pAPE7, ATCCdeposit no. PTA-981. It is another specific object of the invention toprovide a transformed organism, Corynebacterium glutamicum ATCC21799comprising pAPE18, ATCC deposit no. PTA-978.

It is another specific object of the invention to provide analpha-ketobutyrate producing nonhuman organism, comprising one or morecopies of a transgene comprising at least one nucleotide sequenceencoding catabolic threonine dehydratase and, additionally one or moretransgenes comprising at least one nucleotide sequence encoding one ormore enzymes involved in L-threonine biosynthesis, wherein saidtransformed nonhuman organism synthesizes alpha-ketobutyrate, saidsynthesis being greater than that of a corresponding non-transformednonhuman organism.

It is another specific object of the invention to provide a plant, orplant part thereof comprising one or more copies of a transgenecomprising at least one nucleotide sequence encoding catabolic threoninedehydratase.

It is another specific object of the invention to provide a plant, orplant part thereof, wherein said plant or said part thereof, comprisesone or more copies of a transgene comprising at least one nucleotidesequence encoding catabolic threonine dehydratase, wherein said plant orplant part thereof retains more threonine dehydratase enzyme activityduring, or after, contacting herbicide than a corresponding plant, orplant part thereof, contacted with said herbicide, which does notcomprise one or more copies of a transgene comprising at least onenucleotide sequence encoding catabolic threonine dehydratase.

It is another specific object of the invention to provide a method ofproducing a plant, or part thereof, wherein said plant, or plant partthereof, is contacted with one or more copies of a transgene comprisingat least one nucleotide sequence encoding catabolic threoninedehydratase, and wherein said plant, or part thereof, after saidcontacting comprises detectable transgene.

It is another specific object of the invention to provide a method ofusing the plant, or plant part thereof, wherein said plant, or plantpart thereof, is cultivated in the presence of an herbicide and whereingrowth of said plant, or said part thereof, is greater than the growthof a corresponding plant, or plant part thereof, contacted with saidherbicide, which does not comprise one or more copies of a transgenecomprising at least one nucleotide sequence encoding catabolic threoninedehydratase.

It is another specific object of the invention to provide a method ofenhancing resistance to an herbicide in a plant, or plant part thereof,said method comprising introducing into said plant, or plant partthereof, one or more copies of a transgene comprising at least onenucleotide sequence encoding catabolic threonine dehydratase.

Further object and advantages of the present invention will be clearfrom the description that follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Aspartate-derived amino acids pathway. Asp, aspartic acid; hom,homoserine; thr, threonine; met, methionine; lys, lysine; ile,isoleucine; val, valine; leu, leucine; alpha-KB, alpha ketobutyrate;AHB, acetohydroxybutyric acid; PYR, pyruvate; AL, acetolactate; AHAS,acetohydroxyacid synthase; TD, threonine dehydratase.

FIG. 2. Plasmid maps. NG2 rep, ORF from the NG2 replicon permittingplasmid replication in both E. coli and C. glutamicum; P_(trc), trcpromoter from pTrc99a; lacI, ORF encoding lac repressor from pTrc99a;Km^(R), kanamycin resistance gene from pEP2; Nsil/PstI denotes positionof hybrid NsiI and PstI sites resulting from ligation.

FIG. 3. Sensitivity of threonine dehydratases expressed inCoryne-bacterium glutamicum ATCC 21799 to varying isoleucineconcentrations.

FIGS. 4A, 4B. Culture of C. glutamicum ATCC 21799 in batch reactor ondefined medium. Kinetics of growth, substrate consumption (A), and aminoacid production (B). IPTG, arrow indicates point at which IPTG was addedto the culture. In this culture, addition of IPTG did not lead to anincrease in threonine dehydratase (not shown).

FIGS. 5A, 5B, 5C. Culture of C. glutamicum ATCC 21799 (pAPE13) in batchreactor on defined medium. Kinetics of growth, substrate consumption(5A), amino acid production (5C) and production of threonine dehydratase(5B). IPTG, arrow indicates point at which IPTG was added to the culturemedium.

FIGS. 6A, 6B, 6C. Culture of C. glutamicum ATCC 21799 (pAPE7) in batchreactor on defined medium. Kinetics of growth, substrate consumption(6A), amino acid production (6C) and production of catabolic threoninedehydratase (6B). IPTG, point at which IPTG was added to the culturemedium.

FIGS. 7A, 7B. Amino acids (7A) and carbon balance (7B) for the threestrains calculated from final fermentation titers. Lysine and isoleucinecarbon was multiplied by ⅔ to account for pyruvate contribution in theirrespective branches. Alanine carbon was multiplied by ¾ to account forthe economy of 1 mole of CO₂ comparatively to the amino acids of theaspartate-derived pathway.

FIG. 8: Plasmid Map of pAPE18. Plasmid carrying tdcB gene under controlof the trc promoter (Ptrc); a deregulated homoserine dehydrogenase(hom^(dr)); and the homoserine kinase gene (thrB) under control of thetac promoter (Ptac); kanR=kanamycin resistance gene; rep=NG2 rep gene.

DEFINITIONS

In the description that follows, a number of terms used in recombinantDNA (rDNA) are extensively utilized. In order to provide a clear andconsistent understanding of the specification and the claims, thefollowing definitions are provided.

Catabolic threonine dehydratase. As used herein, “catabolic threoninedehydratase” refers to threonine dehydratase enzymes which are directedto the breakdown of threonine in the nonhuman organism. Catabolicthreonine dehydratase enzymes catalyze the conversion of threonine intoalpha-ketobutyrate and ammonia. Catabolic threonine dehydratase is alsoknown as threonine deaminase. Catabolic threonine dehydratases are notinhibited by isoleucine to the same extent as are the biosyntheticthreonine dehydratase enzymes and retain more of their activity even asthe cells accumulate isoleucine. In the invention, “catabolic threoninedehydratase” refers to catabolic threonine dehydratase (E.C.4.2.1.19;formerly E.C.4.2.1.16) as well as to a catabolic threonine dehydrataseisolated and purified from any source, which is encoded by the same, orsubstantially identical, nucleotide sequence as catabolic threoninedehydratase (E.C.4.2.1.19).

Control sequence. As used herein, “control sequence” refers to a DNAsequence or sequences necessary for the expression or regulation(transcriptional or translational) of an operably linked coding sequencein a particular nonhuman organism. The control sequences which aresuitable include, for example, a promoter, optionally an operatorsequence, a ribosome binding site, and a transcription terminator. Manyother sequences such as enhancers, silencers and promoters are known inthe art. See, for example, U.S. Pat. Nos. 4,980,285; 5,631,150;5,759,828; 5,888,783 and, 5,919,670.

Feedback insensitive or feedback resistant. As used herein, the termsare used to denote enzymes (or the genes encoding them) that maintainthe same or similar primary metabolic activity associated with a“wild-type” or native enzyme but whose regulatory properties have beenmodified (through directed mutation or random mutation) or whoseenzymatic regulation is markedly different from that of the originalenzymes(s). For example, while threonine dehydratase encoded by ilvA hasmuch reduced activity in the presence of high levels of isoleucine, tdcBencodes a feedback insensitive threonine dehydratase that maintains moreof its enzymatic activity in the presence of isoleucine. Another exampleis that the homoserine dehydrogenase encoded by hom can be inhibited bythreonine, whereas the homoserine dehydrogenase encoded by hom^(dr) (amodified version of the same gene) retains much more of its activity inthe presence of this metabolite.

Genetic elements. As used herein, “genetic elements” refers to definednucleic acids (generally DNA or RNA) having expressible coding sequencesfor products such as proteins, apoproteins, or antisense nucleic acidconstructs, which can perform or control pathway enzymatic functions.The expressed proteins can function as enzymes, repress or derepressenzyme activity, or control expression of enzymes. The nucleic acidsencoding these expressible sequences can be either chromosomal, e.g.integrated into a nonhuman organism's chromosome by homologousrecombination, transposition, or some other method, or extrachromosomal(episomal), e.g. carried by plasmids, cosmids, etc. Genetic elementsinclude control elements. Many other genetic elements are known in theart. See, for example, U.S. Pat. Nos. 4,980,285; 5,631,150; 5,759,828;5,888,783 and, 5,919,670.

Genetic manipulation. As used herein, the term “genetic manipulation”refers to the purposeful alteration of polynucleotide sequences eitherby in vitro techniques, in vivo techniques, or a combination of both invitro and in vivo techniques. “Genetic manipulation” includes theintroduction of heterologous polynucleotide sequences into nonhumanorganisms, either into the chromosome or as extra-chromosomallyreplicating elements, the alteration of chromosomal polynucleotidesequences, the addition and/or replacement of transcriptional and/ortranslational regulatory signals to chromosomal or plasmid encodedgenes, and the introduction of various insertion, deletion andreplacement mutations in genes of interest. Methods for in vitro and invivo genetic manipulations are widely known to those skilled in the art.See, for example, Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd Ed., Cold Spring Harbor Press (1989) and U.S. Pat. Nos.4,980,285; 5,631,150; 5,759,828; 5,888,783 and, 5,919,670.

Operably linked. As used herein, “operably linked” refers to ajuxtaposition such that the normal function of the components can beperformed. Thus, a coding sequence “operably linked” to controlsequences refers to a configuration wherein the coding sequences can beexpressed under the control of these sequences. Such control may bedirect, that is, a single gene associated with a single promoter, orindirect, as in the case where a polycistronic transcript is expressedfrom a single promoter. See, for example, U.S. Pat. Nos. 4,980,285;5,631,150; 5,707,828; 5,759,828; 5,888,783 and, 5,919,670, and Sambrooket al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold SpringHarbor Press (1989).

Over-expression and Over-Production. As used herein, “over-expression”refers to gene expression. Genes and gene products can be overexpressed.Such gene products include RNAs, proteins and enzymes. On the otherhand, “overproduce” refers to cellular products that accumulate,especially cell products that are to be harvested for some specific use.Thus proteins, materials (such as polymers), and metabolites (such asamino acids) are overproduced. Proteins may be either overexpressed (ifreferring to the control of gene expression) or overproduced (ifreferring to the accumulation of the proteins). By “over production” ofL-isoleucine, it is intended that a nonhuman organism “over producing”L-isoleucine produces more molecules of L-isoleucine for each nonhumanorganism under a given set of growth conditions than a similar nonhumanorganism not “over producing” L-isoleucine.

Plant. As used herein, the term “plant” includes whole plants, plantorgans (e.g., leaves, stems, flowers, roots, etc.), seeds and plantcells and progeny of same. The class of plants which can be used in themethod of the invention is generally as broad as the class of higherplants amenable to transformation techniques, including angiosperms(monocotyledonous and dicotyledonous plants), as well as gymnosperms. Itincludes plants of a variety of ploidy levels, including polyploid,diploid, haploid and hemizygous. See, U.S. Pat. Nos. 5,942,662;5,990,390; and 5,994,622.

Promoter. As used herein, the term “promoter” has its art-recognizedmeaning, denoting a portion of a gene containing DNA sequences thatprovide for the binding of RNA polymerase and initiation oftranscription. Promoter sequences are commonly, but not always, found inthe 5′ non-coding regions of genes. Sequence elements within promotersthat function in the initiation of transcription are often characterizedby consensus nucleotide sequences. Useful promoters include constitutiveand inducible promoters. Many such promoter sequences are known in theart. See, for example, U.S. Pat. Nos. 4,980,285; 5,631,150; 5,707,828;5,759,828; 5,888,783; 5,919,670, and, Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989).Other useful promoters include promoters which are neither constitutivenor responsive to a specific (or known) inducer molecule. Such promotersmay include those that respond to developmental cues (such as growthphase of the culture or stage of cell differentiation), or environmentalcues (such as pH, osmoticum, heat, or cell density). A heterologouspromoter is a promoter which is not naturally linked to the gene.Heterologous promoters may be from the same or different species. Forexample, a heterologous promoter may be a promoter from the sameorganism as the gene but naturally found linked to a different gene.

As used herein, a “plant promoter” is a promoter capable of initiatingtranscription in plant cells. “Plant promoter” has its art-recognizedmeaning, denoting a portion of a gene containing DNA sequences thatprovide for the binding of RNA polymerase and initiation oftranscription. For example, for overexpression, a plant promoterfragment may be employed which will direct expression of the gene in alltissues of a regenerated plant. Such promoters are “constitutive”promoters and are active under most environmental conditions and statesof development or cell differentiation. Examples of constitutivepromoters include the cauliflower mosaic virus (CaMV) 35S transcriptioninitiation region, the 1′- or 2′-promoter derived from T-DNA ofAgrobacterium tumafaciens, and other transcription initiation regionsfrom various plant genes known to those of skill. Alternatively, theplant promoter may an “inducible” promoter. Examples of environmentalconditions that may effect transcription by inducible promoters includeanaerobic conditions, elevated temperature, or the presence of light.Examples of promoters under developmental control include promoters thatinitiate transcription only in certain tissues, such as fruit, seeds,flowers, or ovules. Examples include the AP2 promoter, a promoter fromthe ovule-specific BEL1 gene promoter. See, for example, U.S. Pat. Nos.5,942,662; 5,990,390; and 5,994,622.

Transformed nonhuman organisms. As used herein, the term “transformednonhuman organisms” includes the primary transformed subject cell andits transformed progeny. The nonhuman organism may be prokaryotic oreukaryotic. Thus “transformants” or “transformed cells” includes theprimary subject cell, transformed with the transgene, and culturesderived therefrom, without regard for the number of transfers. It isalso understood that all progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations and/ormodifications. Mutant progeny which have the same functionality asscreened for in the originally transformed cell are included. Wheredistinct designations are intended, it will be clear from the context.See, for example, U.S. Pat. Nos. 4,980,285; 5,631,150; 5,707,828;5,759,828; 5,888,783; 5,919,670, and, Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press (1989).

Transgene. As used herein, the term “transgene” when used in referenceto polynucleotide sequences, refers to polynucleotide sequences notnaturally present in a cell. Thus the term “transgene” includes, forexample, the promoter of gene A operably joined to structural gene B,when A and B genes are from the same organism, as well as the case inwhich a polynucleotide sequence of one species is transferred to a cellof a different species (or strain). The term “transgene” also includesclones of transgenes which have been so modified. See, U.S. Pat. Nos.4,980,285; 5,631,150; 5,707,828; 5,759,828; 5,888,783 and, 5,919,670.

Transgenic Plant. As used herein, the term “transgenic plant” refers toplants having a genome which has been augmented by at least oneincorporated DNA sequence. Such DNA sequences include but are notlimited to genes which are not normally present, DNA sequences notnormally transcribed into RNA or translated into a protein(“expressed”), or any other genes or DNA sequences which are introducedinto the non-transformed plant. Included genes are genes which maynormally be present in the non-transformed plant but which one desiresto either genetically engineer or alter the expression. The genome oftransgenic plants of the present invention will haste been augmentedthrough the stable introduction of the transgene, or. the introducedtransgene will replace an endogenous sequence. See U.S. Pat. No.5,994,622. The term may additionally include “transient” forms of geneexpression to alter plants, i.e., without permanently altering thegenome. Such methods may be accomplished by virus-based gene expressionvectors to introduce genes episomally. Episomally maintained genesaffect gene expression but do not alter the genome.

Vector. As used herein, the term “vector” refers to a polynucleotidesequence suitable for transferring transgenes into a host cell. Hostcells may be plant cells, prokaryotic cells or eukaryotic cells. Theterm may include plasmids, mini-chromosomes, phage, naked DNA and thelike. See, for example, U.S. Pat. Nos. 4,980,285; 5,631,150; 5,707,828;5,759,828; 5,888,783 and, 5,919,670, and, Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989).

DETAILED DESCRIPTION OF THE INVENTION

The disclosed invention relates to nonhuman organisms which express acatabolic threonine dehydratase.

An object of the present invention is to provide an efficient andcost-effective method for synthesizing L-isoleucine by enhancing thecapability of a nonhuman organism, such as bacterium, to produceL-isoleucine. It is a further object of the present invention to providemethods for constructing transformed nonhuman organisms, and thetransformed nonhuman organisms, thereby constructed. The transformednonhuman organisms over-produce L-isoleucine. The transformed nonhumanorganisms also over-express heterologous catabolic threoninedehydratase. In one embodiment, the nonhuman organism used in theinvention belongs to the genus Corynebacterium. In a preferredembodiment, the nonhuman organism is Corynbacterium glutamicum. In aparticularly preferred embodiment, the nonhuman organism isCorynbacterium glutamicum ATCC 21799. In one embodiment, the transformednonhuman organism is of the genus Escherichia. In another embodiment,the transformed nonhuman organism is Escherichia coli. In a preferredembodiment, the transgene comprises a tdcB gene from E. coli.

The transformed nonhuman organisms of the present invention can secretethe synthesized L-isoleucine into the culture medium. This goal isachieved through modification of the metabolism of a desired nonhumanorganism by introducing and expressing desired transgenes. This goal isalso achieved by further modification of the metabolism of certain genesendogenous to such nonhuman organisms in their native state.

It is an object of the invention to provide a method for producingL-isoleucine comprising growing a transformed nonhuman organism underconditions that provide for synthesis of L-isoleucine, wherein saidnonhuman organism comprises one or more copies of a transgene comprisingat least one nucleotide sequence encoding catabolic threoninedehydratase, wherein said L-isoleucine is synthesized by saidtransformed nonhuman organism, said synthesis being greater than that ofa corresponding non-transformed nonhuman organism, and recovering saidL-isoleucine from said culture media in which said transformed nonhumanorganism was cultured.

In one embodiment, the transformed nonhuman organism additionallyover-produces one or more of L-lysine, L-methionine, L-leucine,L-valine, L-threonine, L-aspartic acid, or homoserine, wherein saidproduction is greater than that of a corresponding, nontransformednonhuman organism. In one embodiment, the transformed nonhuman organismis of the genus Corynebacterium. In another embodiment, the nonhumanorganism is Corynebacterium glutamicum. In a preferred embodiment, thenonhuman organism is Corynebacterium glutamicum is ATCC 21799.

In one embodiment, the transgene comprises a tdcB gene from E. coli. Inanother embodiment, the transgene comprises a tdcB gene encodingcatabolic threonine dehydratase (E.C.4.2.1.19). In another embodiment,the transgene comprises a catabolic threonine dehydratase gene fromSalmonella typhimurium. In a preferred embodiment, the nonhuman organismis transformed with pAPE7. In a highly preferred embodiment, thenonhuman organism is Corynebacterium glutamicum and the transgenecomprises a tdcB gene from E. coli.

In one embodiment of the invention, conditions that provide forsynthesis of L-isoleucine comprise supplementation of culture media withone or more amino acids or amino acid precursors. In another embodiment,one or more amino acids or amino acid precursors is one or more ofL-methionine, L-leucine, L-valine, L-threonine, L-lysine, L-asparticacid, glycine, L-alanine and homoserine.

In another embodiment of the method for producing L-isoleucine, thenonhuman organism additionally comprises one or more transgenescomprising at least one nucleotide sequence encoding one or more enzymesinvolved in L-isoleucine biosynthesis. In a preferred embodiment, one ormore enzymes involved in L-isoleucine biosynthesis is one or more ofhomoserine dehydrogenase, homoserine kinase, acetohydroxy acid synthase,aspartokinase, aspartate, β-semialdehyde dehydrogenase, threoninesynthase, acetohydroxy acid isomeroreductase, dihydroxy aciddehydratase, feedback insensitive forms of any of the preceedingenzymes, and any combination thereof.

In one embodiment of the method for producing L-isoleucine, the methodcomprises growing an L-isoleucine producing nonhuman organism comprisingone or more copies of a transgene comprising at least one nucleotidesequence encoding catabolic threonine dehydratase, wherein said nonhumanorganism synthesizes L-isoleucine, said synthesis being greater thanthat of a corresponding nonhuman organism which does not comprise one ormore copies of a transgene comprising at least one nucleotide sequenceencoding catabolic threonine dehydratase and recovering saidL-isoleucine from said culture media in which said L-isoleucineproducing nonhuman organism was cultured. In a preferred embodiment ofthe method the nonhuman organism has been genetically manipulated tooverproduce one or more of L-lysine, L-methionine, L-leucine, L-valine,L-threonine, L-aspartic acid, and homoserine prior to the introductionof said nucleotide sequence encoding catabolic threonine dehydrataseinto said nonhuman organism. In a highly preferred embodiment, thenonhuman organism comprises hom^(dr) and thrB and overproducesthreonine.

It is another object of the invention to provide an L-isoleucineproducing nonhuman organism comprising one or more copies of a transgenecomprising at least one nucleotide sequence encoding catabolic threoninedehydratase, wherein said non human organism synthesizes L-isoleucine,said synthesis being greater than that of the correspondingnon-transformed nonhuman organism. In one embodiment, the nonhumanorganism over-produces L-lysine, L-methionine, L-leucine, L-valine,L-threonine, L-aspartic acid, and homoserine prior to the introductionof said one or more copies of said transgene. In another embodiment, thetransgene comprises a catabolic threonine dehydratase gene from E. colior from Salmonella typhimurium. In a preferred embodiment, the transgenecomprises a tdcB gene encoding catabolic threonine dehydratase(E.C.4.2.1.19). In one embodiment, the nonhuman organism is of the genusCorynebacterium. In another embodiment, the nonhuman organism isCorynebacterium glutamicum. In a preferred embodiment, the nonhumanorganism Corynebacterium glutamicum is ATCC 21799. In one embodiment,the nonhuman organism is of the genus Escherichia. In anotherembodiment, the nonhuman organism is Escherichia coli.

In another embodiment of the invention, the transformed nonhumanorganism additionally comprises one or more transgenes comprising atleast one nucleotide sequence encoding one or more enzymes involved inL-isoleucine biosynthesis. In a preferred embodiment, one or moreenzymes involved in L-isoleucine biosynthesis is one or more ofhomoserine dehydrogenase, homoserine kinase, acetohydroxy acid synthase,aspartokinase, aspartate β-semialdehyde dehydrogenase, threoninesynthase, acetohydroxy acid isomeroreductase, dihydroxy aciddehydratase, feedback insensitive forms of any of the preceedingenzymes, and any combination thereof.

It is another object of the invention to provide an L-isoleucineproducing nonhuman organism comprising one or more copies of a transgenecomprising at least one nucleotide sequence encoding catabolic threoninedehydratase, wherein said nonhuman organism synthesizes L-isoleucine,said synthesis being greater than that of a corresponding nonhumanorganism which does not comprise one or more copies of a transgenecomprising at least one nucleotide sequence encoding catabolic threoninedehydratase. In one embodiment, the nonhuman organism has beengenetically manipulated to overproduce one or more of L-lysine,L-methionine, L-leucine, L-valine, L-threonine, L-aspartic acid, andhomoserine prior to the introduction of said nucleotide sequenceencoding catabolic threonine dehydratase into said nonhuman organism. Ina preferred embodiment, the nonhuman organism comprises hom^(dr) andthrB and overproduces threonine.

It is a further object of the invention to provide Corynebacteriumglutamicum ATCC 21799 transformed with pAPE7, ATCC deposit no. PTA-981.It is another object of the invention to provide Corynebacteriumglutamicum ATCC 21799 transformed with pAPE18, ATCC deposit no. PTA-978.

It is a further object of the invention to provide an alpha-ketobutyrateproducing nonhuman organism, comprising one or more copies of atransgene comprising at least one nucleotide sequence encoding catabolicthreonine dehydratase, and, additionally one or more transgenescomprising at least one nucleotide sequence encoding one or more enzymesinvolved in L-threonine biosynthesis, wherein said transformed nonhumanorganism synthesizes alpha-ketobutyrate, said synthesis being greaterthan that of a corresponding non-transformed nonhuman organism. In oneembodiment of the invention, the nonhuman organism further synthesizesone or more polymers selected from the group consisting ofpolyhydroxyalkanoates (PHAs), polyhydroxybutyrate (PHB), andpoly-hydroxybutyrate-co-valerate (PHBV), and wherein said polymers areaccumulated intracellularly or secreted.

It is another object of the invention to provide a plant, or plant partthereof, comprising one or more copies of a transgene comprising atleast one nucleotide sequence encoding catabolic threonine dehydratase.

It is another object of the invention to provide a plant, or plant partthereof, wherein said plant or said part thereof, comprises one or morecopies of a transgene comprising at least one nucleotide sequenceencoding catabolic threonine dehydratase, wherein said plant or plantpart thereof, retains more threonine dehydratase enzyme activity aftercontacting herbicide than a corresponding plant, or plant part thereof,contacted with said herbicide, which does not comprise one or morecopies of a transgene comprising at least one nucleotide sequenceencoding catabolic threonine dehydratase. In one embodiment, the plant,or part thereof, is selected from leaves, roots, stems, flowers andflower parts, seeds, pollen, cells and calli. In one embodiment, theplant, or part thereof, retains resistance to a herbicide wherein saidherbicide comprises an L-isoleucine analog. In a preferred embodiment,the plant, or part thereof, produces alpha-ketobutyrate and/orisoleucine before contact with, in the presence of, and after saidcontact with said herbicide.

It is another object of the invention to provide a method of producing aplant, or part thereof, wherein said plant, or plant part thereof, iscontacted with one or more copies of a transgene comprising at least onenucleotide sequence encoding catabolic threonine dehydratase, andwherein said plant, or part thereof, after said contacting, comprisesdetectable transgene.

It is another object of the invention to provide a method of using aplant, or plant part thereof, wherein said plant, or plant part thereof,is cultivated in the presence of an herbicide and wherein growth of saidplant, or said part thereof, is greater than the growth of acorresponding plant, or plant part thereof. contacted with saidherbicide, which does not comprise one or more copies of a transgenecomprising at least one nucleotide sequence encoding catabolic threoninedehydratase.

It is another object of the invention to provide a method of enhancingresistance to an herbicide in a plant, or plant part thereof, saidmethod comprising introducing into said plant, or plant part thereof,one or more copies of a transgene comprising at least one nucleotidesequence encoding catabolic threonine dehydratase.

It is further an object of the invention to provide a method for theproduction of L-isoleucine by utilizing a novel metabolic pathway asabove, wherein said pathway modifies the pathways of L-isoleucinebiosynthesis and/or metabolism. In this invention, the pathway ismodified so that the transformed nonhuman organism now producesL-isoleucine by a redirection of the carbon flux from the lysine pathwayto the isoleucine pathway.

It is further an object of the invention to provide a method for theproduction of L-isoleucine by augmenting the preexisting L-isoleucinepathway. The production of L-isoleucine is accomplished by theintroduction and overexpression of the genes coding for catabolicthreonine dehydratase into an L-isoleucine producing nonhuman organism.In another embodiment of the invention, the L-isoleucine producingnonhuman organism additionally overproduces L-lysine.

It is further an object of the invention to provide a method for theproduction of L-isoleucine using the transformed nonhuman organism asabove, the method using the altered L-isoleucine pathway above, suchpathway being altered further by inactivating, using chemically inducedmutagenesis or gene disruption of one or more genes encoding any of theenzymes involved in the biosynthesis of L-amino acids. In one embodimentof the invention, the inactivated genes are involved in the biosyntheticreactions which produce L-isoleucine.

In another object of the invention, over production of L-isoleucine isachieved by inserting a transgene which comprises a nucleotide sequencecoding for an enzyme which is utilized on the route of biosynthesis ofan L-amino acid product into a first vector, and inserting a secondtransgene, which comprises a nucleotide sequence which codes for anenzyme different from said first enzyme on the route of biosynthesis ofsaid L-amino acid into a second vector. The vectors are then introducedinto a strain of a nonhuman organism to transform said strain which iscapable of producing said L-amino acid, wherein the enzymes are highlyrate determining enzymes for the biosynthesis of said L-amino acid.Further, insertion of the first and second transgenes may optionally befollowed by insertion of one or more additional transgenes wherein theadditional transgenes are also highly rate determining enzymes for thebiosynthesis of said L-amino acid.

Any strain of nonhuman organism capable of producing L-amino acids isuseful in the practice of this invention. Such strains include strainslacking a catabolic threonine dehydratase, strains expressingineffective levels of catabolic threonine dehydratase enzymes underspecific conditions (enzyme expression under anaerobic conditions, butlittle or no expression under aerobic conditions, such as in E. coli,for example), strains expressing low levels of catabolic threoninedehydratase enzymes under typical growth conditions and strainsexpressing high levels of catabolic threonine dehydratase.

The first two enzymes in the threonine to isoleucine pathway (threoninedehydratase and acetohydroxyacid synthase) have been found to beimportant in isoleucine biosynthesis. Threonine dehydratase is sensitiveto feedback inhibition by isoleucine and therefore this can be alimiting factor for the improvement of isoleucine biosynthesis. AHAS(acetohydroxyacid synthase) is also feedback inhibited by isoleucine butit has been shown to be highly inducible in the presence of itssubstrate, alpha-ketobutyrate (colón, G. E., et al., Appl. Microbiol.Biotechnol. 43:482-488 (1995); Eggeling, L., et al., Appl. Microbiol.Biotechnol. 25:346-351 (1987)). Therefore threonine dehydratase has beenpreferred as a target for metabolic engineering to increase carbon fluxinto the isoleucine pathway.

The tdcB gene of E. coli encoding catabolic threonine dehydratase hasbeen cloned and inserted in an expression vector for C. glutamicum. ThetdcB gene was expressed in two different strains of Corynebacteriumglutamicum, AS019-E12 and ATCC 21799. In vitro enzymatic assays showedthat the catabolic threonine dehydratase expressed in Corynebacteriumglutamicum retained its insensitivity to isoleucine. A concentration of200 mM isoleucine resulted in only a 40% inhibition of the catabolicthreonine dehydratase whereas just 15 mM isoleucine was able tocompletely inhibit the native threonine dehydratase in the ATCC 21799strain. Accordingly, Morbach S., et al., Appl. Environ. Microbiol.61:4315-4320 (1995), found a complete inhibition of the endogenousthreonine dehydratase (encoded by ilvA) with a concentration of 5 mMisoleucine in Corynebacterium glutamicum MH20-22B. Some authors haveobtained deregulated threonine dehydratase by generating mutations inthe ilvA gene (Hashiguchi, K., et al., Biosci. Biotechnol. Biochem.61:105-108 (1997); Mockel, B., et al., Mol. Microbiol. 13:833-842(1994)). Theirmutatedthreonine dehydratase V323A had 22% activity at 50mM isoleucine (Morbach, S., et al., Appl. Environ. Microbiol.61:4315-4320(1995)) whereas the catabolic threonine dehydratase of E.coli has still 70-80% activity at an identical isoleucine concentration.These results show the potential for using the catabolic threoninedehydratase of E. coli in Corynebacterium.

To determine whether expressing the catabolic threonine dehydratase(tdcB gene) had any greater benefit in isoleucine production whencompared to overexpression of the native enzyme, encoded by ilvA, twooverexpression vectors were constructed, one carrying the ilvA gene, theother carrying the tdcB gene. These plasmids were introduced into alysine producing strain, ATCC 21799, a strain whose feedback-resistantaspartokinase permits high lysine production (Jetten, M. S. M., et al.,Appl. Microbiol. Biotechnol. 43:76-82 (1995)). The results showed that20-fold overexpression of the ilvA gene product, threonine dehydratase,led to a 4-fold increase in the isoleucine production (0.2 g·l⁻¹). Thislow yield can be explained by a reduction in activity of the threoninedehydratase due to the feedback inhibition by isoleucine and/or by theinhibiting effect of leucine added in the medium. ATCC 21799 is aleucine auxotroph requiring the inclusion of leucine in the medium.However, the excess leucine may be decreasing the activity ofacetohydroxy acid synthase (AHAS), the second enzyme of the isoleucinepathway, via feedback inhibition (Eggeling, L., et al., Appl. MicrobiolBiotechnol. 25:346-351 (1987)). AHAS has been indeed found to beinhibited by leucine and valine, and its expression is repressedmultivalently by all three branched-chain amino acids (Eggeling, L., etal., Appl. Microbiol. Biotechnol. 25:346-351 (1987); Tsuchida, T. &Momose, H., Agric. Biol. Chem. 39:2193-2198 (1975)). In comparison,15-fold overexpression of the catabolic threonine dehydratase led to asignificant 50-fold increase in the isoleucine production (2.5 g·l⁻¹) atthe expense of lysine production (1.4 g·l⁻¹). This observationeliminates the possibility that leucine-inhibition of AHAS enzyme wasresponsible for low isoleucine yield in the ilvA overexpressing strain,since excess leucine was included in the tdcB expressing cultures aswell.

In order to determine the distribution of the carbon throughout theaspartate-derived amino acid pathway in the different strains, aminoacid and carbon balances in the amino acid pathways were compared in thethree strains (FIG. 7). The carbon balances have been corrected toaccount for incorporation of 1 mole of pyruvate into the lysine andisoleucine pathways and for the economy of 1 mole of CO₂ for thesynthesis of alanine, and thus represent only derivatives of the carbonskeleton of aspartate. The three strains produced comparable amount ofamino acids. In the ilvA overexpressing strain, only 5% of the carbonavailable for the aspartate-derived amino acids pathway was directed toisoleucine and 75% into lysine. A higher concentration of homoserine andalanine was produced in this strain. While applicants do not wish to belimited by any single explanation, this result can be explained by thefact that an increase of homoserine concentration at the expense oflysine (as a result of ilvA overexpression) leads to a higheravailability of pyruvate that can be converted into alanine.

The balances show that in the strain carrying the plasmid containing thetdcB gene the carbon flux has been redirected from the lysine pathwaythrough the isoleucine pathway. In this strain, 70% of the carbonavailable for the aspartate-derived amino acids pathway has beenconverted into isoleucine. As a comparison, colón, G. E., et al., Appl.Microbiol. Biotechnol. 43:482-488 (1995), found that 80% of the carbonavailable for the aspartate-derived amino acids pathway has beenconverted to isoleucine in an ilvA overexpressing ATCC 21799 strain, butin that case they used a threonine overproducing strain as the host forilvA overexpression. Thus, this host strain also overexpressed aderegulated homoserine dehydrogenase (hom^(dr)) and the homoserinekinase (thrB). The present study demonstrates that overexpression oftdcB alone in a lysine producing strain is sufficient to driveisoleucine overproduction to a level comparable to that demonstratedwith a three-gene system (hom^(dr), thrB, ilvA).

Growth kinetics showed that the strain carrying the plasmid containingthe tdcB gene grew more slowly than the wild type or the ilvAoverexpressing strains. This could be explained by a depletion of one ormore amino acids, a consequence of the redirection of the carbon fluxafter expression of the tdcB gene. The inhibitory effect ofalpha-ketobutyrate on the growth of Corynebacterium glutamicum has beenalready reported (Eggeling, L., et al., Appl. Microbiol. Biotechnol.25:346-351 (1987)). Addition of a mixture of amino acids from a caseinhydrolysate to the medium reestablished optimal growth of the strainexpressing the tdcB gene. Further investigations showed thatspecifically an addition of valine or methionine led to a partialrecovery of the growth of this strain, 80 and 86% recovery respectively.The addition of these two amino acids together gave a total growthrecovery. Thus the overexpression of the feedback-resistant threoninedehydratase draws carbon away from the valine pathway. The isoleucineand valine pathways compete for pyruvate (FIG. 1) as a substrate. Theenzyme AHAS catalyzes the second reaction of the isoleucine pathway bycondensing alpha-ketobutyrate and pyruvate and also catalyzes the firstreaction of the valine pathway by condensing two molecules of pyruvate.In contrast to E. coli, it has been shown that no isoenzymes of AHASexist in C. glutamicum (Eggeling, L., et al., Appl. Microbiol.Biotechnol. 25:346-351 (1987)). The same authors showed that this enzymehas a higher V_(max) and a 3-fold higher affinity for alpha-ketobutyratethan for pyruvate. Moreover, the inhibitory effect of alpha-ketobutyrateon the growth of C. glutamicum has been already reported (Eggeling, L.,et al., Appl. Microbiol. Biotechnol. 25:346-351 (1987)). Similarly, thisinhibition can be overcome by addition of valine plus leucine. Thus theincrease of the catabolic threonine dehydratase leads to an increase ofthe amount of available alpha-ketobutyrate. This alpha-ketobutyrate thenout competes pyruvate leading to more acetohydroxybutyrate synthesisthan acetolactate synthesis. The end result is that the valine supplyfalls short (excess leucine has been supplied in the medium). Similarly,overcoming the growth inhibition of the strain comprising pAPE7 byaddition of methionine in the medium could be explained by the fact thatthe overexpression of the catabolic threonine dehydratase directs carbonflux preferentially from homoserine to threonine at the expense of themethionine pathway leading also to a reduced supply of methionineprecursors.

Expression of the tdcB gene under aerobic conditions was accomplished byintroducing a heterologous promoter. The regulatory properties of thisenzyme were not altered. The catabolic threonine dehydratase in E. coliis normally inhibited by high concentrations of pyruvate and some otheralpha-keto acids, but this inhibition can be completely overcome byincreased levels of AMP (Feldman, D. A. & Datta, P., Biochem.14:1760-1767 (1975)). While these effectors may be operating inCorynebacterium, it is clear that there is sufficient threoninedehydratase activity in the tdcb-carrying strain to promote isoleucineproduction. As a result, the production of isoleucine has been increasedby a factor 50, and 70% of the carbon available for the lysine pathwayhas been directed into the isoleucine pathway. Expressing this enzyme instrains with different genetic backgrounds, such as, for example, athreonine overproducing strain, would provide an additional benefit interms of isoleucine production.

Threonine dehydratase (sometimes called “threonine deaminase”) is anenzyme that catalyzes the conversion of threonine intoalpha-ketobutyrate and ammonia. This enzyme is a critical component inthe pathway toward isoleucine biosynthesis and has been the subject ofresearch in the last several years for the purposes of creatingisoleucine overproducing strains of bacteria.

In addition to the threonine dehydratases involved in isoleucinebiosynthesis, Escherichia coli and Salmonella typhimurium possesscatabolic threonine dehydratases dedicated to the breakdown of threoninein the cell (Bhadra, R. & Datta, P., Biochem. 179:1691-1699 (1978)).While these catabolic enzymes also generate alpha-ketobutyrate, it isclear that regulation of their expression and activity is very differentfrom regulation of the biosynthetic forms of the enzymes. Specifically,whereas the “biosynthetic” threonine dehydratases (such as those encodedby the ilvA genes of Corynebacterium and E. coli) that are involved inisoleucine biosynthesis are inhibited by high levels of isoleucine,catabolic threonine dehydratases are not inhibited by isoleucine to thesame extent and retain more of their activity even as cells accumulateisoleucine.

In addition to the aforementioned capability of producing L-isoleucine,a nonhuman organism of the present invention may have the knowncharacteristics which are effective in enhancing its capability ofproducing an amino acid, for example, various nutrient requirements,resistance to drugs, sensitivity to drugs, and drug dependence, orcharacteristics wherein a gene promoting the biosynthesis of an aminoacid is amplified by means of gene engineering. Methods of geneamplification in order to increase copy number are known in the art.See, for example, U.S. Pat. Nos. 4,980,285; 5,631,150; 5,707,828;5,759,828; 5,888,783 and, 5,919,670, and, Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989).

The method of production of L-isoleucine of the invention can beperformed by culturing the nonhuman organism of the present invention ina liquid medium to allow L-isoleucine to be produced. The L-isoleucinethus produced is accumulated in the liquid medium, and collected fromthis liquid medium. In another embodiment, the L-isoleucine-producingnonhuman organism of the present invention is also used in theproduction of L-lysine, any other L-amino acid or any amino acidprecursor.

In the L-isoleucine producing method of the present invention which isthe cultivation of the L-isoleucine-producing nonhuman organism, thecollection and purification of L-isoleucine from the culture medium maybe performed in a manner similar to the conventional fermentation methodwherein an amino acid is produced using a nonhuman organism. A mediumused for culture may be either a synthetic medium or a natural medium,so long as the medium includes a carbon source and a nitrogen source andminerals and, if necessary, appropriate amounts of nutrients which thenonhuman organism requires for growth. The carbon source may includevarious carbohydrates such as glucose and sucrose, and various organicacids. Depending on the mode of assimilation of the used nonhumanorganism, alcohol including ethanol, methanol and glycerol may be used.As the nitrogen source, various ammonium salts such as ammonia andammonium sulfate, other nitrogen compounds such as amines, a naturalnitrogen source such as peptone, soybean-hydrolysate and yeast extractare used. Many other sources are known in the art. As minerals,potassium monophosphate, magnesium sulfate, sodium chloride, ferroussulfate, manganese sulfate, calcium carbonate, etc., are used. Suchmethods are known in the art. See, for example, U.S. Pat. Nos.4,980,285; 5,631,150; 5,707,828; 5,759,828; 5,888,783 and, 5,919,670,and, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Press (1989).

After cultivation, solids such as cells can be removed from the culturemedium by gravity, centrifugation and/or membrane filtration, and thenthe target L-isoleucine can be collected and purified by ion-exchange,ion exclusion, concentration, chromatographic and crystallizationmethods. The methods of 5 recovery, isolation, purification andcrystallization are by any means known to those of skill in the art.Further, L-isoleucine is produced either continuously, or, in batchculture. The culture media containing the synthesized L-isoleucineover-producing nonhuman organism is analyzed at any time before, duringor after the culturing for the presence and/or amount of L-isoleucineproduced. Methods of collecting and purifying amino acids are known inthe art. See, for example, U.S. Pat. Nos. 4,980,285; 5,707,828;5,759,828; 5,888,783 and, 5,919,670, and, Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989).

The mechanism for transforming the host cell to direct carbon flow intothe divergent pathway preferably involves the insertion of geneticelements including control sequences and sequences coding for catabolicthreonine dehydratase. Regardless of the exact mechanism utilized, it iscontemplated that the expression of these enzymatic activities will beeffected or mediated by the transfer of recombinant genetic elementsinto the host cell. In a preferred embodiment of the invention, therecombinant genetic element is a transgene wherein the transgene is acatabolic threonine dehydratase gene from E. coli or a catabolicthreonine dehydratase gene from Salmonella typhimurium. It is furtherenvisioned that the catabolic threonine dehydratase gene may be from anyprokaryotic or eukaryotic organism. Further, the prokaryotic organism iscryophilic, mesophilic or thermophilic.

The genetic elements of the present invention can be introduced into anonhuman organism by vectors such as plasmids, cosmids, phages or viralvectors that mediate transfer of the genetic elements into a nonhumanorganism. These vectors can include an origin of replication along withcis-acting control elements that control replication of the vectors andthe genetic elements carried by the vectors. Selectable markers can bepresent on the vectors to aid in the identification of nonhumanorganisms into which the genetic elements have been introduced. Forexample, selectable markers can be genes that confer resistance toparticular antibiotics such as tetracycline, ampicillin,chloramphenicol, kanamycin, or neomycin. Other selectable markers knownin the art are suitable in the practice of the invention. See, forexample, U.S. Pat. Nos. 4,980,285; 5,631,150; 5,707,828; 5,759,828;5,888,783 and, 5,919,670, and, Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989).

The expression of a gene is primarily directed by its own promoter,although other genetic elements including optional expression controlsequences such as repressors and enhancers can be included to controlexpression or derepression of coding sequences for proteins,apoproteins, or antisense constructs.

In addition, DNA constructs can be generated whereby the gene's naturalpromoter is replaced with an alternative promoter to increase expressionof the gene product. Promoters can be either constitutive or inducible.A constitutive promoter controls transcription of a gene at a constantrate during the life of a cell, whereas an inducible promoter's activityfluctuates as determined by the presence (or absence) of a specificinducer, or in response to developmental or environmental cues. Forexample, control sequences can be inserted into wild type nonhumanorganisms to promote overexpression of selected enzymes already encodedin the genome of the nonhuman organism, or alternatively can be used tocontrol synthesis of extrachromosomally (episomal) encoded enzymes. Thenucleic acid construct is introduced into the nonhuman organism bycontacting the nonhuman organism with the nucleic acid construct. Typesof contacting include transduction, transformation or transfection, orother mechanisms, such as electroporation or microinjection, known tothose of skill in the art. See, for example, U.S. Pat. Nos. 4,980,285;5,631,150; 5,707,828; 5,759,828; 5,888,783 and, 5,919,670, and, Sambrooket al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold SpringHarbor Press (1989).

Catabolic threonine dehydratases that are much less sensitive toisoleucine inhibition will be useful in any technology that requires athreonine dehydratase to function under conditions where isoleucine maybe present in high concentrations, high enough to inhibit all or some ofthe activity normally carried out by the “biosynthetic” enzyme, or underconditions where other inhibitors are present, such as amino acidanalogues. In many biological systems, the native, anabolic threoninedehydratase is sensitive to feedback inhibition by isoleucine ormolecules that resemble isoleucine (e.g., synthetic analogs ofisoleucine). Thus a drug or other compound may act by inhibiting thisenzyme, thereby starving a cell for isoleucine, alpha-ketobutyrate orsome other threonine catabolite. Similarly, a herbicide that mimics theeffect of isoleucine and inhibits this enzyme would starve the cell,tissue or whole plant of these compounds. Because they are lesssensitive to isoleucine-mediated inhibition, catabolic threoninedehydratases such as that encoded by tdcB can be used to produce cells,plants, etc, that are resistant to such drugs or herbicides.Applications of this technology can be envisioned to produce, forexample, herbicide resistant crops and drug resistant cell lines. Suchapplications include prokaryotic, eukaryotic, or in vitro systems wherethreonine is determined by the enzyme in order to supply metabolites orprecursors for other cellular processes or products.

Advances in recombinant DNA technology coupled with advances in planttransformation and regeneration technology have made it possible tointroduce new genetic material into plant cells, plants or plant tissue,thus introducing new traits, e.g., phenotypes, that enhance the value ofthe plant or plant tissue. Thus, there has developed a strong interestin engineering the genome of plants to contain and express foreigngenes, or additional, or modifed versions of native, or endogenous genes(perhaps driven by different promoters) in order to alter the traits ofa plant in a specific manner. See, for example, U.S. Pat. No. 6,005,168.Demonstrations of the production of herbicide tolerant plants (DeBlock,M. et al., EMBO J. 6:2513 (1987)) highlight the potential for cropimprovement. The target crops can range from trees and shrubs toornamental flowers and field crops. “Crop” as used herein can also be aculture of plant tissue grown in a bioreactor as a source for somenatural product. See, for, example, U.S. Pat. No. 5,942,662.

Various methods are known in the art to accomplish the genetictransformation of plants and plant tissues using transgenes. See, forexample, U.S. Pat. Nos. 5,942,662, 5,990,390 and 6,005,168. Theseinclude transformation by Agrobacterium species and transformation bydirect gene transfer. Transformation of plant cells mediated byinfection with Agrobacterium tumefaciens and subsequent transfer of theT-DNA alone have been well documented (Bevan, M. et al., Int. Rev.Genet. 16: 357 (1982)). Other gene transfer procedures have beendeveloped to transform plants and plant tissues without the use of anAgrobacterium intermediate (Koziel et al., Biotechnology 11: 194-200(1993)). In the direct transformation of protoplasts the uptake ofexogenous genetic material into a protoplast may be enhanced by use of achemical agent or electric field. The exogenous material may then beintegrated into the nuclear genome (Paszkowski, J. et al., EMBO J. 3:2717 (1984) and Potrykus, I. et al., Mol. Gen. Genet. 199: 169 (1985)).Polyethylene glycol (PEG)-mediated DNA uptake has been demonstrated inprotoplasts (Lorz et al., 1985). DNA may be introduced into intact plantcells by electroporation (PCT WO 92/12250). Alternatively, exogenous DNAcan be introduced into cells or protoplasts by microinjection (Reich, T.J. et al., Bio/Technology 4: 1001 (1986)). One procedure for direct genetransfer involves bombardment of cells by microprojectiles carrying DNA(Klein, T. M. et al., Nature 327:70 (1987)). Other methods may also beused for introduction of DNA into plant cells, for example, by agitationof cells with DNA and silicon carbide fibers.

Techniques for transforming a wide variety of higher plant species arewell known and described in the technical and scientific literature.See, for example, Weising et al., Ann. Rev. Genet. 22: 421-477 (1988).

Vectors for use in accordance with the present invention may beconstructed to include one or more regulatory elements controlling geneexpression in plants. Such elements include enhancer elements, promoterelements, transcriptional sequences, translational sequences,termination sequences or any other regulatory element known to those ofskill in the art. Examples of enhancer elements useful in the practiceof the invention include the ocs enhancer. Many other promoters usefulin plant tissue expression are known to those of skill in the art.

Promoters which direct specific or enhanced expression in certain planttissues are known to those of skill in the art. These include, forexample, the rbcS promoter, specific for green tissue; the ocs, nos andmas promoters which have higher activity in roots or wounded leaftissue; a truncated (−90 to +8) 35S promoter which directs enhancedexpression in roots, an alpha-tubulin gene that directs expression inroots and promoters which direct expression in endosperm. Other knownpromoters include CaMV 35S promoter, CaMV 19S, nos, Adh, sucrosesynthase, alpha-tubulin, actin, cab, PEPCase or those associated withthe R gene complex. Tissue specific promoters such as root cellpromoters and tissue specific enhancers are also contemplated to beparticularly useful, as are inducible promoters such as ABA- andturgor-inducible promoters.

Novel promoters or enhancers which are homologous or tissue specific(e.g., root-, collar/sheath-, whorl-, stalk-, earshank-, kernel- orleaf-specific) are envisioned in the practice of the invention. It isalso envisioned that one use of the present invention will be theexpression of a catabolic threonine dehydratase gene in atissue-specific manner.

Vectors will also include a 3′ end DNA sequence that acts as a signal toterminate transcription and allow for the poly-adenylation of theresultant mRNA. Known 3′ elements are those from the nopaline synthasegene of Agrobacterium tumefasciens, the terminator for the T7 transcriptfrom the octopine synthase gene of Agrobacterium tumefasciens, and the3′ end of the protease inhibitor I or II genes from potato or tomato.Regulatory elements such as Adh intron 1, sucrose synthase intron or TMVomega element, may further be included where desired.

A leader sequence may also be employed in the practice of the invention.Preferred leader sequences are contemplated to include those whichinclude sequences predicted to direct optimum expression of the attachedtransgene, i.e., to include a preferred consensus leader sequence whichmay increase or maintain mRNA stability and prevent inappropriateinitiation of translation.

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype and thus the desired phenotypesuch as increased isoleucine expression and/or alpha-ketobutyratesynthesis. Such regeneration techniques rely on hormonal manipulation ina tissue culture growth medium, typically relying on a biocide and/orherbicide marker which has been introduced together with the desirednucleotide sequences.

Plant regeneration from cultured protoplasts is described in Evans etal., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture,pp. 124-176, MacMillilan Publishing Company, New York, 1983; andBinding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRCPress, Boca Raton, 1985. Regeneration can also be obtained from plantcalli, explants, organs, or parts thereof. Such regeneration techniquesare described generally in Klee et al., Ann. Rev. of Plant Phys. 38:467-486 (1987). See, U.S. Pat. No. 5,994,662.

Genes that confer resistance to herbicides are well known in the art.For example, several herbicides, such as an imidazalinone or asulfonylurea, are known to inhibit the growing point or meristem.Exemplary genes conferring resistance to these herbicides encode mutantALS and AHAS enzymes (Lee et al., EMBO J. 7:1241 (1988); Miki et al.,Theor. Appl. Genet. 80: 449 (1990), and U.S. Pat. No. 5,952,553).Another herbicide, glyphosate, has resistance imparted by mutant5-enolpyruvyl-3-phosphoshikimate synthase (EPSP) encoded by the aroAgene. U.S. Pat. No. 4,940,835 discloses the nucleotide sequence of aform of EPSP which can confer glyphosate resistance. The nucleotidesequence of the mutant aroA gene is disclosed in U.S. Pat. No.4,769,061. Nucleotide sequences of glutamine synthetase genes whichconfer resistance to herbicides such as L-phosphinothricin are disclosedin European patent application No.0333 033, and U.S. Pat. No. 4,975,374.The nucleotide sequence of a phosphinothricin-acetyl-transferase gene isprovided in European patent application No. 0 242 246. De Greef et al.(Bio/Technology 7: 61 (1989)) describe the production of transgenicplants that express chimeric bar genes coding for phosphinothricinacetyl transferase activity. Genes conferring resistance to phenoxyproprionic acids and cyclohexones, such as sethoxydim and haloxyfop, arethe Accl-S1, Accl-S2 and Accl-S3 genes, (Marshall et al., Theor. Appl.Genet. 83: 435 (1992)). See also U.S. Pat. No. 6,005,168. The deh geneencodes the enzyme dalaphon dehalogenase and confers resistance to theherbicide dalapon. The bxn gene codes for a specific nitrilase enzymethat converts bromoxynil to a non-herbicidal degradation product. Othergenes conferring resistance to other herbicides are known in the art.See, for example, U.S. Pat. No. 5,990,390.

The presence of the transgene can be detected by any method known tothose of skill in the art. Such methods, include, but are not limited toamplification techniques, such as PCR, for example, and, Southernhybridization techniques and modifications thereof, such as dot or slothybridization, for example.

In any biological system where breakdown products of threonine arerequired for useful biological process, the tdcB gene may be used byitself, in place of, or to supplement, existing enzymes because of itsinsensitivity to feedback regulation. One application of this technologyis the production of biopolymers. Metabolism of threonine toalpha-ketobutyrate is an important step in synthesizing usefulprecursors for biopolymer synthesis. Catabolic threonine dehydrataseconverts threonine to alpha-ketobutyrate. The alpha-ketobutyrate ismetabolized further, for example to useful CoenzymeA derivatives such aspropionyl-CoA, and the subsequent metabolites can be incorporated intopolymers such as polyhydroxyalkanoates (PHAs). This strategy isespecially useful for producing co-polymers such aspoly-(hydroxybutyrate-co-hydroxyvalerate) (PHBVs). If a cell has only a“normal” feedback sensitive, anabolic threonine dehydratase, ittypically cannot synthesize enough of the alpha-ketobutyrate to supplythe needs of polymer synthesis. Applications of this technology can beeasily envisioned for use in both prokaryotic systems (e.g. bacterial)and eukaryotic systems (e.g. plants and fungi). See, for example, U.S.Pat. Nos. 5,958,745; 5,534,432; 5,245,023; 5,480,794; 5,334,520;5,518,907; 5,344,769; 5,135,859; 5,602,321; 5,663,063; 5,250,430 and5,942,660. See, also, Valentin, H. E., et al., Int. J. Biol. Macromol.25(1-3): 303-306 (1999).

The present invention is described in further detail in the followingnon-limiting examples.

EXAMPLES

The following protocols and experimental details are referenced in theexamples that follow.

I. Strains, Plasmids and Media

Bacterial strains and plasmids are listed in Table 1. LB medium or 2xYTmedium (Sambrook, J., et al., Molecular Clonning: A Laboratory Manual,2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989))was used as a standard medium, and a medium containing 40 g·l⁻¹brain-heart infusion, 20 g·l⁻¹ sorbitol and 10 g·l⁻¹ sucrose was used asa recovery broth for electroporated cells. Minimal medium for E. coliwas M9 medium. E. coli AB1255 (Pittard, J. & Adelberg, E. A., J.Bacteriol. 85:1402-1408 (1963)) was obtained from the E. coli GeneticStock Center, Department of Biology, Yale University, New Haven, Conn.06520, courtesy of Barbara Bachman, and minimal medium for this strainwas supplemented with 100 mg·l⁻¹ each of histidine, arginine, andmethionine.

Defined medium for Corynebacterium glutamicum contained the followingingredients (per liter): glucose, 35 g; NaCl, 2 g; citrate (trisodiumsalt, dihydrate), 3 g; CaCl₂.2H₂O, 0.1 g; MgSO₄.7H₂O, 0.5 g;Na₂EDTA.2H₂O, 75 mg; FeSO₄.7H₂O, 50 mg; 100×salt solution, 20 ml;K₂HPO₄, 4 g; KH₂PO₄, 2 g; (NH₄)₂SO₄, 7.5 g; urea, 3.75 g; leucine 0.85g; thiamine, 0.45 mg; biotin, 0.45 mg; pantothenic acid, 4.5 mg (pH7.0). The salt solution contained the following ingredients (per liter):MnSO₄, 200 mg; Na₂B₄O₇.10H2O, 20 mg; (NH₄)₆Mo₇O₂₄.4H₂O, 10 mg;FeCl₃.6H₂O, 200 mg; ZnSO₄.7H₂O CuCl₂.2H₂O, 20 mg (pH 2.0). Whenappropriate, kanamycin (150 mg/liter) andisopropyl-β-D-thiogalactopyranoside (IPTG) (150 mg/l) was used.

For the growth study with amino acid supplements, the defined medium wascomplemented with Bacto® casamino acids (Difco, Detroit, Mich.) at aconcentration of2 g·l⁻¹ or with amino acid (alanine, glycine, methionineor valine) at a concentration of 0.5 g·l⁻¹ each.

TABLE 1 Bacterial strains. AEC^(R) denotes resistance toaminoethylcysteine Strains Genotype or Description Source or ReferenceC. glutamicum restriction deficient Follettie. M.T., et al., Mol.AS019-E12 derivative of C. Microbiol. 2:53-62 (1988) glutamicum AS019 C.glutamicum Lysine-producing strain, ATCC ATCC21799 Leu⁻. AEC^(R), PanE.coli λ⁻, ung-I, relA1, spoT1, Duncan, B.K & Weiss, B., J. BW310 thi-1Bacteriol. 151:750-755 (1982) AB1255 ilvA201 metB1 hisG1 Pittard, J.,and Adelberg, argH1 E.A., J. Bacteriol. 85:1402- 1408 (1963)

II. Cloning

tdcB and ilvA coding regions were amplified by the Polymerase ChainReaction (PCR) (Table 2), from BW310 genomic DNA and from pGC77 (Km^(R),Ap^(R), LacI^(q), hom^(dr), tac::thrB, ilvA)(Table 1), respectively. ThePCR products were cloned into the pCRScript system (Ap^(R))(Stratagene,La Jolla) creating tdcB-pCRscript (Ap^(R), tdcB (no promoter)) andpAPE16 (Ap^(R), irc:ilvA), again respectively. The EcoRI-BamHI fragmentof these pCRscript derivatives were cloned into pTrc99a (Ap^(R),LacI^(q) trc)(Pharmacia, Upsalla, Sweden) to create pAPE5 (Ap^(R),LacI^(q), trc:tdcB) and pAPE17 (Ap^(R), LacI^(q), trc:ilvA),respectively. Subsequently, the NsiI-SalI fragments of pAPES (Ap^(R),LacI^(q), trc:tdcB), pAPE17 (Ap^(R), LacI^(q), trc:ilvA), and pTrc99a(Ap^(R), including the lacI^(q) and P_(trc) element) were subcloned intopEP2 (Km^(R) NG2 rep)(Zhang, Y., et al., J Bacteriol. 176:5718-5728(1994)), which had been cut with PstI and with SalI, to create pAPE7(Km^(R), LacI^(q), trc:tdcB), pAPE13 (Km^(R), LacI^(q), trc:ilvA), andpAPE12 (Km^(R), LacI^(q), trc), all of which could replicate both inCorynebacterium and in E.coli, and which expressed the appropriate geneproduct (or empty control) under control of the trc promoter. Plasmidmaps are shown in FIG. 2.

TABLE 2 PCR products and primers PCR Product Template Description Primer1 Primer 2 tdcB BW310 Coding region for 5′gggaattcggtgtcgg5′ccggtaccccaaac genomic catabolic threonine ttacggttacct3′aagcctaacgtcca 3′ DNA dehydratase from SEQ ID NO: 1 SEQ ID NO: 2 E.coliilvA pGC77 Coding region for 5′ggaattcatgagtgaa 5′ccacgcgtggggcttanabolic threonine acatacgtgtctga 3′ tgcgatcct 3′ dehydratase from C.SEQ ID NO: 3 SEQ ID NO: 4 glutamicum

III. Enzyme Assays

Enzyme assays were performed with cell free crude extracts, prepared bythe following method. Cells were harvested by centrifugation for 10 minat 5000×g at 4° C. and washed with 10 ml of the enzyme assay buffer (100mM Tris-HCl, pH 7.5, containing 20 mM KCl, 5 mM MnSO₄, 0.1 mM EDTA, and2 mM dithiothreitol) (Jetten, M. S. M., et al., Appl. Microbiol.Biotechnol. 41:47-52 (1994)). The cell pellet was resuspended in thesame buffer containing a protease inhibitor cocktail (BoebringerMannheim, Germany) so that the final concentration was 20 g dry cellweight·l⁻¹. Resuspended cells were disrupted with a glass bead mixer(5100 Mixer-Mill, SPEX, Metuchen, N.J.). 2.5 ml of bacterial suspensionwas poured in a frozen steel vial containing 5 g of cold 106 νm-diameterglass beads (Sigma) and one stainless steel ball bearing. The vialscontaining bacterial suspension and glass beads were vigorously shakenat 4° C. using the mixer by 10 cycles of 30 sec shaking separated byrest cycles of one minute. Cell debris were removed by centrifugationfor 20 min at 47000×g at 4° C. The supernatant (crude extract) was thentested for enzyme activity. Protein concentrations were determined bythe method of Bradford (Bradford, M. M., Anal. Biochem. 72:248-254(1976)) with the Bio-Rad (Hercules, Calif.) protein assay kit usingbovine serum albumin as a standard.

For determination of threonine dehydratase activity, the assay mixturecontained 40 mM threonine, 1 mM pyridoxal phosphate, crude extract and100 mM potassium phosphate buffer, pH 8.0 in a final volume of 1 ml. Thereaction was started with the addition of threonine and terminated bythe addition of 1 ml solution containing 1% semicarbazide and 0.9%sodium acetate. After a 15 min incubation at room temperature, theamount of alpha-ketobutyrate formed at various intervals was measuredspectrophotometrically as its semicarbazone derivative at 254 nm (=0.52mmol·cm⁻¹). Relevant standards and controls were carried out in the samemanner. For specific determination of catabolic threonine dehydrataseactivity, the assay mixture contained 20 mM isoleucine in order toinhibit the anabolic threonine dehydratase.

In FIGS. 3-6, the results of the enzymatic assays are expressed in termsof relative expression of threonine dehydratase compared to wild typeexpression as the ratio between the specific activity of the enzyme of astrain and the specific activity of the wild type. Each assay wasreplicated five times, and the results were reproducible within 15%.

IV. Fermentations

Starter cultures of Corynebacterium glutamicum were prepared bytransferring one colony from LB agar plates to 5 ml of LB medium. Thesecultures were incubated for two days at 30° C. and 200 rpm. 500ml-Erlenmeyer flasks containing 50 ml of defined medium were inoculatedwith 1 ml of the starter culture. Cultures were incubated at 30° C. and200 rpm for 30 hours. These flask cultures were used as a 5% (v/v)inoculum for 2 liters of defined medium in a 4-liter Chemap CMF100reactor (Alfa-Laval Chemap, Switzerland). The culture was grown at 30°C. with an aeration rate of 0.45 VVM and an agitation rate of 1500 rpm.The pH was maintained at 7.5 with ammonium hydroxide and hydrochloricacid solutions. Fermentations were carried out twice for each strain.

V. Determination of Biomass, Sugars, Organic Acids and Amino Acids

During the fermentation, samples were collected and centrifuged at10,000×g, 4° C., for 10 minutes. For biomass determination, cell dryweight was determined gravimetrically.

For determination of glucose, organic acids and amino acids, sampleswere collected and filtered through 0.2 μm Acrodisc® filters (GelmanSciences, Ann Arbor, USA). Sugars and organic acid concentrations weredetermined by HPLC (Hewlett Packard model 1050, Waldbronn, Germany)using an Aminex® HPX-87H column (Bio-Rad, Hercules, USA). Sampleanalysis was performed at 40° C. using 5 mM sulfuric acid as the mobilephase at a flow rate of 0.6 ml·min⁻¹. The detection of sugars wasperformed with a refractive index detector (Hewlett Packard model 1047A)and the detection of organic acids with a UV detector (Hewlett Packardmodel 1050). Results from replicate measurements of glucose werereproducible within 5%.

Amino acids were analyzed as ortho-phthaldialdehyde derivatives byreversed-phase chromatography using a C 18 AminoQuant column with aHewlett Packard series 1050 high-pressure liquid chromatography (HPLC)system (Hewlett Packard, Waldbronn, Germany). Amino acid determinationswere reproducible within 5% in replicate assays.

Example 1 Overexpression of tdcB in E. coli and in C. glutamicum

Enzyme assays for threonine dehydratase activity were conducted on crudeextracts from an E. Coli strain (AB1255) which cannot produce anabolicthreonine dehydratase (encoded by the ilvA gene) and the same straincarrying the pAPE5 plasmid containing the tdcB gene. Even in thepresence of very high concentrations of isoleucine (up to 47 mM), theAB1255 (pAPE5) extracts showed consistent activity at about 80 μmolproduct·min⁻¹·mg protein⁻¹, while AB1255 (pTrc99a) control extracts hadno measurable activity at any isoleucine concentration. These resultsindicate that the tdcB gene product can be overexpressed in aerobicconditions in E. coli while still retaining function.

In order to test expression of the catabolic threonine dehydratase inCorynebacterium, the trc:tdcB fusion was subcloned from pAPE5 into theexpression vector pAPE12, and this plasmid (pAPE7) was expressed in C.glutamicum AS019-E12. Whereas a control strain of C. glutamicumAS019-E12 carrying pAPE12 had no detectable catabolic threoninedehydratase activity, the strain carrying pAPE7 produced 9 μmolproduct·min⁻¹·mg protein⁻¹. Although the activity measured in crudeextracts of this strain was about 10-fold lower than that in E. coli,the activities are actually proportional to the differences in plasmidcopy number in these two bacteria, in that pAPE7 is expected to have a10-fold lower copy number in Corynebacterium than pAPE5 has in E. coli.These results show that tdcB is expressed in the heterologous species.

Cultures of Corynebacterium glutamicum ATCC 21799 comprising plasmidpAPE13(ilvA) and Corynebacterium glutamicum ATCC 21799 comprisingplasmid pAPE7 (tdcB) were deposited under the terms of the BudapestTreaty on the International Recognition of the Deposit of Nonhumanorganisms for Purposes of Patent Procedure at the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209.The deposits of the cultures of the mutants were accepted by thatDepository under the terms of that Treaty. The deposit ofCorynebacterium glutamicum ATCC 21799 comprising plasmid pAPE7 (tdcB)was assigned deposit number PTA-98 1. The deposit of Corynebacteriumglutamicum ATCC 21799 comprising plasmid pAPE12(Km^(R), LacI^(q), trc)was assigned deposit number PTA-979. The deposit of Corynebacteriumglutamicum ATCC 21799 comprising plasmid pAPE13(ilvA) was assigneddeposit number PTA-980. The deposit of Corynebacterium glutamicum ATCC21799 comprising plasmid pAPE18 (tdcB, hom^(dr), Ptrc, thrB, Ptac,Km^(R), rep) was assigned deposit number PTA-978.

Example 2 Isoleucine Sensitivity of the Threonine Dehydratases

In order to confirm the insensitivity to isoleucine of catabolicdehydratase expressed in a lysine producing strain of C. glutamicum,threonine dehydratase activities were measured in crude extracts ofstrains ATCC21799 and ATCC21799 carrying pAPE7 at differentconcentrations of isoleucine (FIG. 3). Anabolic threonine dehydratase ofthe wild type strain ATCC21799 was totally inhibited by an isoleucineconcentration of 15 mM. On the other hand, catabolic threoninedehydratase expressed in strain ATCC 21799 (pAPE7) was much lesssensitive to isoleucine. It retained 60% of its original activity evenat a concentration of 200 mM isoleucine.

Example 3 Fermentation Results

In order to determine whether or not there was an advantage to using thecatabolic threonine dehydratase for the production of isoleucine,fermentations were performed with Corynebacterium glutamicum ATCC 21799as a control, and two derivatives of this strain carrying either thepAPE13 plasmid comprising the trc:ilvA fusion or carrying the pAPE7plasmid comprising the trc:tdcB fusion.

I. Fermentation with the Wild Type Strain

C. glutamicum ATCC 21799 was grown twice in defined medium in a 4-literreactor. IPTG (150 mg/l) was added when the biomass reached 1.5 g celldry weight·l⁻¹. Kinetics of growth, substrate consumption, threoninedehydratase specific activity and amino acid production are shown FIG.4. This strain grew exponentially with a specific growth rate of 0.3 h⁻¹and a glucose to biomass conversion yield of 0.53 g cell dry weight·gglucose⁻¹. The basal level of threonine dehydratase stayed constantaround 1 μmol alpha-ketobutyrate·min⁻¹·mg protein⁻¹ during thefermentation course. The strain produced mainly lysine at finalconcentrations of 3.9 and 4.2 g·1⁻¹, in the two fermentations. Aconcentration of isoleucine of 50 mg·l⁻¹ was detected at the end of bothcultures. Oxygenation of the cultures being sufficient, neither lactatenor acetate was detected during the fermentation.

II. Fermentation with the ilvA Overexpressing Strain

C. Glutamicum ATCC 21799 harboring pAPE13 was grown twice in definedmedium in a 4 liter reactor. As before, IPTG (150 mg/l) was added whenbiomass reached 1.5 g cell dry weight·l⁻¹(FIG. 5). The specific growthrate and the glucose to biomass conversion yield of this strain wereidentical to the wild type strain. The addition of IPTG resulted in a20-fold increase in the level of threonine dehydratase. ATCC 21799(pAPE13) still produced mainly lysine at final concentrations of 3.2 and2.9 g·l⁻¹ in the two fermentations. The concentration of isoleucinereached a value of 0.2 g·l⁻¹ at the end of both fermentations.

III. Fermentation with the tdcB Overexpressing Strain

C. glutamicum ATCC 21799 harboring pAPE7 was grown twice in definedmedium in a 4 liter reactor under the same conditions of agitation,aeration and IPTG addition as the two former strains (FIG. 6). Thespecific growth rate (0.22 h⁻¹) and the glucose to biomass conversionyield (0.46 g.g⁻¹) of this strain were lower than those obtained withthe wild type strain and ilvA4 overexpressing strain. The addition ofIPTG resulted in the synthesis of catabolic threonine dehydratase whichreached a 15-fold higher concentration than that of the original enzyme.Catabolic threonine dehydratase activity remained high during the courseof the fermentation. As a result, ATCC 21799 (pAPE7) produced 2.5 g·l⁻¹isoleucine and 1.3 g·l⁻¹ lysine in the first fermentation, 2.3 g·l⁻¹isoleucine and 1.3 g·l⁻¹ lysine in the second fermentation.

Example 4 Growth Study with Amino Acid Supplements

To investigate the slow growth of the tdcB expressing strain, C.glutamicum ATCC 21799 containing pAPE7 and pAPE13 were cultured inconical flasks with defined medium supplemented with amino acids (Table3). The tdcB expressing strain, when cultured on the defined mediumsupplemented with a mixture of amino acids from a casein hydrolysate(casamino acids), recovered to an optimal growth from 0.17 he withoutcasamino acids to 0.29 h⁻¹ with casamino acids. This rate was comparableto the growth of the wild type and ilvA overexpressing strains (0.30h⁻¹). In order to determine which specific amino acid was limiting,causing the slow growth rate in the tdcB expressing strain, the definedmedium was supplemented with specific amino acids depending on theirpotential interaction with the isoleucine pathway. Valine and methioninewere chosen to test due to their direct connection with the isoleucinepathway, and alanine and glycine for their indirect connection throughthe use of pyruvate, which is also a substrate for the isoleucinepathway.

TABLE 3 Growth study of ilvA and tdcB expressing C. glutamicum strainson defined medium supplemented with amino acids and containing 150 mg/lIPTG Specific growth rate (h⁻¹) Defined medium ATCC 21799 ATCC 21799supplemented with pAPE13ilvA pAPE7tdcB — 0.30 0.17 casamino acids 0.300.29 Valine nd^(a) 0.24 Methionine nd^(a) 0.26 Alanine nd^(a) 0.17Glycine nd^(a) 0.17 Valine + Methionine nd^(a) 0.29 (^(a)Not Determined)

Results showed that the addition of valine or methionine led to anincrease of the specific growth rate (to 0.24 and 0.26 h⁻¹,respectively). The addition of both valine and methionine to the definedmedium resulted in a growth rate comparable to that seen when casaminoacids were added to the medium (0.29 h⁻¹). Alanine or glycine alone wereunable to restore growth rate (0.17 h⁻¹) of the tdcB expressing strain.

Having now fully described the present invention in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that same can beperformed by modifying or changing the invention with a wide andequivalent range of conditions, formulations and other parametersthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

4 1 28 DNA Artificial Sequence misc_feature Primer 1 gggaattcggtgtcggttac ggttacct 28 2 28 DNA Artificial Sequence misc_feature Primer2 ccggtacccc aaacaagcct aacgtcca 28 3 30 DNA Artificial Sequencemisc_feature Primer 3 ggaattcatg agtgaaacat acgtgtctga 30 4 24 DNAArtificial Sequence misc_feature Primer 4 ccacgcgtgg ggctttgcga tcct 24

What is claimed is:
 1. A method for producing L-isoleucine comprising:(a) growing a transformed nonhuman organism under conditions thatprovide for synthesis of L-isoleucine, wherein said nonhuman organismcomprises one or more copies of a transgene comprising at least onenucleotide sequence of a tdcB gene encoding catabolic threoninedehydratase (E.C.4.2.1.19); wherein said L-isoleucine is synthesized bysaid transformed nonhuman organism, said synthesis being greater thanthat of a corresponding non-transformed nonhuman organism; and (b)recovering said L-isoleucine from said culture media in which saidtransformed nonhuman organism was cultured.
 2. The method of claim 1,wherein said transformed nonhuman organism additionally over-producesone or more of L-lysine, L-methionine, L-leucine, L-valine, L-threonine,L-aspartic acid, and homoserine, wherein said production is greater thanthat of a corresponding, nontransformed nonhuman organism.
 3. The methodof claim 1, wherein the transformed nonhuman organism is of the genusCorynebacterium.
 4. The method of claim 1, wherein the transformednonhuman organism is of the genus Escherichia.
 5. The method of claim 1,wherein said transgene comprises a tdcB gene from E. coli.
 6. The methodof claim 1, wherein said transgene comprises a catabolic threoninedehydratase gene from Salmonella typhimurium.
 7. The method of claim 1,wherein said nonhuman organism is transformed with pAPE7.
 8. The methodof claim 1, wherein said nonhuman organism is Corynebacterium glutamicumand said transgene comprises a tdcB gene from E. coli.
 9. The method ofclaim 1, wherein said conditions that provide for synthesis ofL-isoleucine comprise supplementation of said culture media with one ormore amino acids or amino acid precursors.
 10. The method of claim 1 forproducing L-isoleucine, wherein said nonhuman organism additionallycomprises one or more transgenes comprising at least one nucleotidesequence encoding one or more enzymes involved in L-isoleucinebiosynthesis.
 11. The method of claim 3, wherein said nonhuman organismis Corynebacterium glutamicum.
 12. The method of claim 4, wherein thetransformed nonhuman organism is Escherichia coli.
 13. The method ofclaim 9, wherein said one or more amino acids or amino acid precursorsis one or more of L-methionine, L-leucine, L-valine, L-threonine,L-lysine, L-aspartic acid, glycine, L-alanine and homoserine.
 14. Themethod of claim 11, wherein said Corynebacterium glutamicum is ATCC21799.
 15. The transformed nonhuman organism of claim 10, wherein saidone or more enzymes involved in L-isoleucine biosynthesis is one or moreof homoserine dehydrogenase, homoserine kinase, acetohydroxy acidsynthase, aspartokinase, aspartate β-semialdehyde dehydrogenase,threonine synthase, acetohydroxy acid isomeroreductase, dihydroxy aciddehydratase, and feedback insensitive forms thereof.
 16. A method forproducing L-isoleucine comprising: (a) growing an L-isoleucine producingnonhuman organism comprising one or more copies of a transgenecomprising at least one nucleotide sequence of a tdcB gene encodingcatabolic threonine dehydratase (E.C.4.2.1.19) wherein said non humanorganism synthesizes L-isoleucine, said synthesis being greater thanthat of a corresponding nonhuman organism which does not comprise one ormore copies of a transgene comprising at least one nucleotide sequenceencoding catabolic threonine dehydratase; and (b) recovering saidL-isoleucine from said culture media in which said L-isoleucineproducing nonhuman organism was grown.
 17. The method of claim 16wherein said nonhuman organism has been genetically manipulated tooverproduce one or more of L-lysine, L-methionine, L-leucine, L-valine,L-threonine, L-aspartic acid, and homoserine prior to the introductionof said nucleotide sequence encoding catabolic threonine dehydrataseinto said nonhuman organism.
 18. The method of claim 17 wherein saidnonhuman organism comprises hom^(dr) and thrB and overproducesthreonine.
 19. An L-isoleucine producing nonhuman organism comprisingone or more copies of a transgene comprising at least one nucleotidesequence of a tdcB gene encoding catabolic threonine dehydratase(E.C.4.2.1.19), wherein said nonhuman organism synthesizes L-isoleucine,said synthesis being greater than that of a correspondingnon-transformed nonhuman organism.
 20. The nonhuman organism of claim19, wherein said nonhuman organism over-produces one or more ofL-lysine, L-methionine, L-leucine, L-valine, L-threonine, L-asparticacid, and homoserine prior to the introduction of said one or morecopies of said transgene.
 21. The nonhuman organism of claim 19, whereinsaid nonhuman organism additionally over-produces one or more ofL-lysine, L-methionine, L-leucine, L-valine, L-threonine, L-asparticacid, and homoserine.
 22. The nonhuman organism of claim 19, whereinsaid transgene comprises a catabolic threonine dehydratase gene from E.coli or from Salmonella typhimurium.
 23. The nonhuman organism of claim19, wherein said transgene comprises a tdcB gene encoding catabolicthreonine dehydratase (E.C. 4.2.1.19).
 24. The nonhuman organism ofclaim 19, wherein said nonhuman organism is of the genusCorynebacterium.
 25. The nonhuman organism of claim 19, wherein saidnonhuman organism is of the genus Escherichia.
 26. The transformednonhuman organism of claim 19, wherein said transformed nonhumanorganism additionally comprises one or more transgenes comprising atleast one nucleotide sequence encoding one or more enzymes involved inL-isoleucine biosynthesis.
 27. The nonhuman organism of claim 24,wherein said nonhuman organism is Corynebacterium glutamicum.
 28. Thenonhuman organism of claim 25, wherein said nonhuman organism isEscherichia coli.
 29. The transformed nonhuman organism of claim 26,wherein said one or more enzymes involved in L-isoleucine biosynthesisis one or more of homoserine dehydrogenase, homoserine kinase,acetohydroxy acid synthase, aspartokinase, aspartate β-semialdehydedehydrogenase, threonine synthase, acetohydroxy acid isomeroreductase,dihydroxy acid dehydratase, and feedback insensitive forms thereof. 30.The nonhuman organism of claim 27, wherein said Corynebacteriumglutamicum is ATCC
 21799. 31. An L-isoleucine producing nonhumanorganism comprising one or more copies of a transgene comprising atleast one nucleotide sequence of a tdcB gene encoding catabolicthreonine dehydratase (E.C.4.2.1.19), wherein said nonhuman organismsynthesizes L-isoleucine, said synthesis being greater than that of acorresponding nonhuman organism which does not comprise one or morecopies of a transgene comprising at least one nucleotide sequenceencoding catabolic threonine dehydratase.
 32. The nonhuman organism ofclaim 31 wherein said nonhuman organism has been genetically manipulatedto overproduce one or more of L-lysine, L-methionine, L-leucine,L-valine, L-threonine, L-aspartic acid, and homoserine prior to theintroduction of said nucleotide sequence encoding catabolic threoninedehydratase into said nonhuman organism.
 33. The nonhuman organism ofclaim 32 wherein said nonhuman organism comprises hom^(dr) and thrB andoverproduces threonine.
 34. Corynebacterium glutamicum ATCC21799comprising pAPE7, ATCC deposit no. PTA-981.
 35. Corynebacteriumglutamicum ATCC21799 comprising pAPE18, ATCC deposit no. PTA-978.
 36. Aplant, or plant part thereof, comprising one or more copies of atransgene comprising at least one nucleotide sequence of a tdcB geneencoding catabolic threonine dehydratase, wherein said plant or plantpart thereof retains more catabolic threonine dehydratase enzymeactivity after contacting herbicide than a corresponding plant, or plantpart thereof, contacted with said herbicide, which does not comprise oneor more copies of a transgene comprising at least one nucleotidesequence encoding catabolic threonine dehydratase.
 37. The plant, orplant part thereof, of claim 36, wherein said plant part is selectedfrom leaves, roots, stems, flowers and flower parts, seeds, pollen,cells and calli.
 38. The plant, or plant part thereof, of claim 36wherein said herbicide comprises an L-isoleucine analog.
 39. The plant,or plant part thereof, of claim 36 wherein said plant, or plant partthereof, produces alpha-ketobutyrate and/or isoleucine before contactwith, in the presence of, and after said contact with said herbicide.40. A method of increasing plant growth comprising: cultivating theplant, or plant part thereof, of claim 36 in the presence of anherbicide, wherein growth of said plant, or said plant part thereof, isgreater than the growth of a corresponding plant, or plant part thereof,contacted with said herbicide, which does not comprise one or morecopies of a transgene comprising at least one nucleotide sequenceencoding catabolic threonine dehydratase.
 41. A method of enhancingresistance to a herbicide in a plant, or plant part thereof, said methodcomprising introducing into said plant, or plant part thereof, one ormore copies of a transgene comprising at least one nucleotide sequenceof a tdcB gene encoding catabolic threonine dehydratase (E.C.4.2.1.19),thereby enhancing resistance to a herbicide.